Estrogen promotes seizure activity both in women with epilepsy and in animal models of epilepsy, but the cellular events that underlie estrogen promotion of seizures are not known. It is known that estrogen induces changes in the structure and function of excitatory synapses in the hippocampus that are likely to increase seizure susceptibility. Estrogen increases the density and number of dendritic spines (sites of excitatory synaptic connections) and spine synapses on hippocampal neurons, increases hippocampal neurons' sensitivity to excitatory synaptic input, and increases the divergence of excitatory synaptic input from individual presynaptic cells to multiple postsynaptic cells; these changes in synaptic connectivity are associated with greater susceptibility to hippocampus-dependent behavioral seizures. Knowledge of the mechanism(s) by which these estrogen-induced changes in hippocampal synapses occur is critical to understanding cellular processes leading to synaptic rearrangements that increase seizure susceptibility. We developed a novel system for organotypic slice culture of adult rat hippocampus that models changes in hippocampal circuitry induced by estrogen in vivo. This new in vitro system allows analyses of estrogen regulation of hippocampal circuitry using approaches that are not possible with in vivo experiments. In the proposed studies, we will perform both in vivo manipulations in adult female rats and in vitro manipulations in adult slice cultures to answer three key questions about the mechanisms of estrogen's effects on hippocampal synaptic connectivity: 1) What is the role of subcortical structures, particularly cholinergic neurons in basal forebrain? 2) What type(s) of estrogen receptors are involved? 3) What is the role of disinhibition? The proposed studies will integrate anatomical and electrophysiological measures of estrogen's effects on excitatory and inhibitory synaptic interactions in the hippocampus with behavioral analyses of seizure susceptibility. As such, these studies will reveal new information about the cellular mechanisms by which estrogen-induced changes in synaptic connectivity occur and will rigorously test the association of structural and functional changes in hippocampal synaptic connectivity with seizure susceptibility.